2. in addition to numerous biochemical changes, which in turn alter neuronal function and
viability (Pekny and Nilsson, 2005). Reactive astrocytes express proteins capable of both
beneficial and detrimental effects on parameters of neuronal health, outcomes of which are
dependent upon factors such as the age of the organism and the type and extent of injury
(Sofroniew, 2009). Since astrogliosis can result in such functionally diverse outcomes on
neuronal survival, a thorough understanding of the signal transduction pathways regulating
astrocyte reactivity may provide insight into normal and pathological astrocytic responses to
injury.
Caspase activation is a widespread event following disease or damage to the brain, not only
present in neurons and microglia, but also observed in astrocytes (Acarin et al., 2007;
Benjelloun et al., 2003; Mouser et al., 2006; Narkilahti et al., 2003; Su et al., 2000; Villapol
et al., 2008). Caspases are cysteine proteases classified as either initiator caspases, including
caspase-2, -8, -9 and -10, or executioner caspases, including caspase-3, -6 and -7. Initiator
caspases proteolytically cleave and activate executioner caspases. In turn, executioner
caspases proteolytically cleave and degrade structural proteins, signaling molecules, and
DNA repair enzymes (Kumar, 2006). While the caspase family is predominantly associated
with the processing of pro-inflammatory cytokines and the execution of apoptosis (Chang
and Yang, 2000), various non-apoptotic functions including modulation of cell proliferation,
differentiation and cell spreading have been described in a variety of cell types
(McLaughlin, 2004; Schwerk and Schulze-Osthoff, 2003). In the brain, the caspase family
have also been implicated in neuronal (Fernando et al., 2005) and glial differentiation
(Oomman et al., 2006), neuronal cytoskeletal remodeling (Acarin et al., 2007; Rohn et al.,
2004), synaptic plasticity (Dash et al., 2000), axon guidance and neurite outgrowth
(Campbell and Holt, 2003; Chan and Mattson, 1999) as well as contributing to neuronal
survival following preconditioning (Garnier et al., 2003; McLaughlin, 2004; Tanaka et al.,
2004).
While some studies attribute caspase activation in astrocytes to glial degeneration (Su et al.,
2000), several studies have also demonstrated that astrocytic expression of active caspases is
not always associated with cell death following ischemic/excitotoxic damage (Acarin et al.,
2007; Duran-Vilaregut et al., 2010; Narkilahti et al., 2003; Villapol et al., 2008; Wagner et
al., 2011) and traumatic brain injury (Beer et al., 2000). Considering that gliosis, not cell
death, is the principal astrocytic response to injury, it is possible that caspase activation in
astrocytes may exert non-apoptotic functions that contribute to astrogliosis.
To investigate a possible non-apoptotic function of caspases in the regulation of astrogliosis,
we examined caspase activity and indices of reactivity in rat astrocyte cultures. Experiments
were conducted in two different culture paradigms. First, we utilized primary cultures of
neonatal rat astrocytes that were exposed to β-amyloid (Aβ), an aggregating peptide that is
associated with astrogliosis in both Alzheimer’s disease brain (Rodriguez et al., 2009) and
culture models. Treatment of astroglial cultures with Aβ typically does not result in cell
death (Kato et al., 1997; Pike et al., 1994) but causes morphological features of gliosis (Abe
et al., 1997; Canning et al., 1993; Hu et al., 1998; Pike et al., 1994; Salinero et al., 1997) as
well as increased glial fibrillary acid protein (GFAP) immunoreactivity (Pike et al., 1994;
Hu et al., 1998) and elevated levels of several cytokines, growth factors, and enzymes
(Araujo and Cotman, 1992; Hu et al., 1998; Pike et al., 1994, 1996a). For comparison, we
also treated cultures with dibutryl cyclic AMP (dBcAMP), a stimulus that induces some
features of astrogliosis (Fedoroff et al., 1984; Le Prince et al., 1991). In our analyses, we
focused on two factors that are established markers of astrogliosis in several paradigms,
basic fibroblast growth factor (FGF-2) and glutamine synthetase (GS) (Eddleston and
Mucke, 1993). Observations from neonatal cultures studies were corroborated using an ex
vivo model of astrogliosis induced in adult animals, an approach demonstrated to retain
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3. biochemical changes associated with the reactive phenotype in culture even after multiple
divisions (Rozovsky et al., 2005; Wu et al., 1998).
2. RESULTS
2.1 In vitro model of astrogliosis
Neonatal astrocyte cultures were treated with either dBcAMP (1 mM), a synthetic analogue
of cAMP, or Aβ peptide 25–35 (Aβ, 25 μM), an aggregating polypeptide implicated in
Alzheimer’s disease. Exposure to treatments for 48 h induced stellation (Fig. 1A–C) and
significantly increased expression of GS (Fig. 1D) and FGF-2 (Fig. 1E). These astrogliosis-
related changes were observed as early as 24 h after exposure to dBcAMP and Aβ and
persisted for at least 7 d (data not shown).
2.2 Non-apoptotic activation of caspases in neonatal astrocyte cultures
To begin evaluating a potential role of caspases in the observed astrogliosis, we assessed
caspase activity in astrocytes cultures treated with dBcAMP or Aβ. In comparison to
vehicle-treated controls, cultures exposed for 48 h to 1 mM dBcAMP or 25 μM Aβ showed
more than a two-fold increase in total caspase activity (Fig. 2A). This increase in caspase
activity was apparent within 24 h and persisted for at least 4 d, although there was no
significant change in vehicle-treated control cultures (data not shown). The observed
elevation in caspase activity was associated with an up-regulation of the cleaved active
fragment of caspase-3, the most common effector caspase in the brain (Fig. 2B). Increases in
total caspase activity and cleavage of caspase-3 associated with dBcAMP- and Aβ-induced
reactivity were modest compared to marked increases observed following treatment of
astrocytes with a toxic concentration of staurosporine (1 μM), a broad kinase inhibitor
established to induce caspase activation and apoptosis in numerous cell types (Bertrand et
al., 1994; Krohn et al., 1998). Importantly, although both 1 mM dBcAMP and 25 μM Aβ
increase caspase activity in astrocytes, this action was not associated with changes in cell
viability. To ensure that caspase activation was not associated with delayed cell death, we
maintained treatment of astrocyte cultures with dBcAMP and Aβ for 4 days before analysis.
Neither dBcAMP nor Aβ induced significant cell death compared to vehicle-treated
astrocytes, with no differences observed in the number of cells labeled with the cell death
marker, ethidium homodimer (Fig. 3A, B) or the release of the enzyme lactate
dehydrogenase (Fig. 3C). The proportion of vehicle-treated cells labeled with ethidium
homodimer was always <5% at all time points. As a positive control, we also assessed
viability in cultures treated with 1 μM staurosporine.
2.3 Caspase inhibitors modulate reactive responses to Aβ and dBcAMP in neonatal
astrocyte cultures
To begin to assess the potential role of the observed increase in caspase activity in regulation
of astrogliosis, we evaluated the effect of a broad-spectrum caspase inhibitor, zVAD, on
expression of GS and FGF-2 as well as morphological stellation. We observed that 1 h
pretreatment of astrocyte cultures with 50 μM zVAD significantly reduced the increases in
GS (Fig. 4A,B) and FGF-2 (Fig. 4C,D) induced by 48 h exposure to 1 mM dBcAMP and 25
μM Aβ. In separate experiments, we found that expression of GS and FGF-2 was also
reduced by a 48 h exposure to 50 μM zVAD initiated 48 h after the addition of dBcAMP and
Aβ (data not shown). In contrast to its effects on GS and FGF-2 expression, zVAD
pretreatment did not affect induction of stellate morphology by dBcAMP and Aβ (Fig. 5A,
B). As a positive control, we also assessed the effects of pretreatment with 10 nM thrombin,
a factor known to reverse stellate morphology of astrocytes (Pindon et al., 1998), and
observed an approximately 75% inhibition of astrocyte stellation in dBcAMP and Aβ treated
cultures (Fig. 5A, B).
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4. To investigate which members of the caspase family may be contributing to observed effect
of the broad spectrum caspase inhibitor, astrocyte cultures were pretreated for 1 h with
synthetic peptide inhibitors specific for caspase-1 (YVAD), caspase-3 (DEVD), caspase-6
(AEVD), caspase-8 (LETD), caspase-9 (LEHD) or caspase-11 (WEHD) prior to inducing
reactivity with 1 mM dBcAMP. Since the inhibitors can show reduced specificity at higher
concentrations (>50 μM), only inhibitory effects on GS and FGF-2 expression observed at
lower concentrations (1, 10 and 30 μM) were considered specific. Specific inhibition of
caspase-3 with DEVD (Fig. 6A, B) and caspase-11 with WEHD (Fig. 6C, D) significantly
attenuated dBcAMP-induced elevations in GS and FGF-2 expression. At higher, less
specific concentrations (> 50 μM), AEVD and LEHD significantly attenuated both GS and
FGF-2 expression, while YVAD attenuated FGF-2 but not GS expression (Fig. 7).
2.4 Caspases contribute to astrogliosis in adult astrocyte cultures
To corroborate the relationship between caspases and astrogliosis observed in cultures of
neonatal astrocytes, we used a second experimental model of hippocampal astrocytes
cultured from kainate-lesioned adult rats and sham-lesioned control rats. We used
immunohistochemical analyses to confirm that kainate induced both neuron loss (neuron-
specific NeuN antibody) and associated astrogliosis (astrocyte-specific GFAP antibody) in
the lesioned animals. As expected, we observed extensive neuron loss confined to the CA3
region of hippocampus (Fig. 8A,B) and robust astrogliosis throughout hippocampus (Fig.
8C,D). Cultures generated from kainate-lesioned and sham-lesioned animals exhibited
similar morphological appearance with predominantly polygonal-shaped cells and an
absence of significant stellation (Fig. 9A,B). Both kainate-lesioned and sham-lesioned
astrocytes cultures were also characterized by low levels of cell death as evidenced by
minimal release of lactate dehydrogenase (Fig. 9C).
There were significant differences between astrocytes cultures generated from kainate-
lesioned and sham-lesioned in terms of both caspase activity and expression of the
astrogliosis markers GS and FGF-2. Cultures from kainate-lesioned animals exhibited an
approximately two-fold increase in caspase activity (Fig. 9D) and elevated levels of the
active, cleaved fragment of caspase-3 (Fig. 9E), relative to cultures from sham-lesioned
animals. Levels of both total caspase activity and active caspase-3 were modest in
comparison to that observed in astrocyte cultures from sham-lesioned animals treated for 24
h with 1 μM staurosporine. Astrocyte cultures from kainate-lesioned animals were also
characterized by increased expression of GS and FGF-2 (Fig. 10), demonstrating a retained
reactive phenotype ex vivo. Treatment for 24 h with the broad-spectrum caspase inhibitor
zVAD significantly attenuated expression of GS and FGF-2 in cultures from kainate-
lesioned rats but not sham-lesioned rats (Fig. 10).
3. DISCUSSION
Following injury, caspase activation in neurons is predominantly associated with neuronal
apoptosis. In contrast, in vivo studies have demonstrated poor correlation between caspase
activation in astrocytes and cell death (Beer et al., 2000; Narkilahti et al., 2003). In the
current study, we demonstrate a functional significance of non-apoptotic activation of
caspases, wherein caspase inhibition modulates astrogliotic responses in vitro. Inhibition of
caspase activity using a broad-spectrum caspase inhibitor partially attenuated upregulation
of two proteins associated with astrogliosis, GS and FGF-2, without altering reactive
morphology. Use of subtype-specific caspase inhibitors suggested functional involvement of
caspases-3 and -11 in specific aspects of astrogliosis. While these findings suggest a key role
of caspase activation in regulating astrogliosis, there are potential limitations to this
conclusion. For instance, specific features of astrogliosis, and consequently the signaling
pathways underlying astrogliosis, are known to vary across different paradigms (Norton et
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5. al., 1992; Wu and Schwartz, 1998). In this context, it is important to note that we observed
similar changes in GS, FGF-2, and caspase activation with different inducing stimuli and
across two different culture paradigms. The implications of this will be discussed in the
context of our broadening understanding of non-apoptotic functions of the caspases in the
brain.
3.1 Evidence for functional involvement of caspase-3 and caspase-11 in astrogliosis
In the current study, caspase activation was observed in the absence of cell death in both in
vitro and ex vivo models of astrogliosis. Consistent with the increase in total caspase
activity, an up-regulation of the cleaved active fragment of caspase-3 was observed. Further,
specific inhibition of caspase-3 attenuated biochemical markers of astrogliosis (GS and
FGF-2). While nuclear translocation of caspase-3 is indicative of its proteolytic activity and
is usually associated with apoptosis (Kamada et al., 2005), previous in vivo studies have
demonstrated nuclear caspase-3 activation in non-apoptotic astrocytes following brain injury
(Acarin et al., 2007; Beer et al., 2000; Benjelloun et al., 2003; Mouser et al., 2006; Su et al.,
2000; Villapol et al., 2008). Caspase-3 has been implicated in the cleavage of structurally
important proteins including vimentin (Byun et al., 2001) and GFAP in astrocytes (Mouser
et al., 2006). While caspase-3 cleavage of structural proteins may be the result of
cytoskeletal dismantling and cell death (Mouser et al., 2006), cleaved GFAP and vimentin
have also been found to co-localize with activated caspase-3 in non-apoptotic astrocytes in
injury models, indicating a potential non-apoptotic role for caspase-3 in astrocyte
cytoskeletal remodeling (Acarin et al., 2007). Interestingly, our findings show that caspase
inhibition did not alter reactive morphology and thus argue that caspase activation alone is
not sufficient for cytoskeletal remodeling associated with hypertrophy and stellation. We
speculate that cytoskeletal changes in astrogliosis reflect contributions from caspase
activation and additional signaling pathways. For example, stellation in cultured astrocytes
treated with dBcAMP or Aβ is dependent upon Na+ and K+ transport across the plasma
membrane (Abe and Saitoh, 2001).
In our in vitro model of astrogliosis, caspase-11 was also implicated in regulating
astrogliosis since specific pharmacological inhibition of caspase-11 significantly reduced
expression of GS and FGF-2. Caspase-11 is predominantly known as an inflammatory
initiator caspase, essential for activation of caspase-1 (Wang et al., 1998). No evidence of
caspase-1 involvement in the astrocyte responses to dBcAMP or Aβ was observed in the
current study since specific inhibition caspase-1 did not alter expression of the assessed
markers of astrogliosis. An alternative role of caspase-11 may be activation of caspase-3,
which has been demonstrated under certain pathological conditions (Kang et al., 2000).
Further investigation is required to determine if a similar such pathway contributes to our
observations.
3.2 Potential mechanisms underlying non-apoptotic caspase activation in astrocytes
Activated caspases are known to regulate several non-apoptotic functions, although the
mechanism(s) underlying these actions remain to be fully elucidated. In some systems,
caspases mediate their non-apoptotic actions via proteolytic activation of transcription
factors. For example, differentiation of erythrocytes involves non-apoptotic caspase-
mediated cleavage and activation of GATA-1 (De Maria et al., 1999). Similarly, skeletal
muscle differentiation involves a crucial role of caspase-3 activation of Mammalian Sterile
Twenty-like kinase (Fernando et al., 2002). Several transcription factors and signaling
molecules that are activated during astrogliosis, including CREB and NF-κB (de Freitas et
al., 2002; Nikaido et al., 2002), are established caspase substrates (Fran∂cois et al., 2000;
Qin et al., 2000) and could be potential down-stream effectors of caspase activation. For
instance, NF-κB, while initially thought to be inactivated upon caspase cleavage, is now
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6. known to be activated even by low levels of caspase activation, which in turn leads to
activation of inflammatory pathways (Lamkanfi et al., 2006). Also, proliferation of reactive
astrocytes following a cortical stab wound is associated with a down-regulation of the cell-
cycle arrest protein p27kip1 (Koguchi et al., 2002). Caspase-mediated cleavage and
inactivation of p27kip1 is essential for proliferation of transformed lymphoid cell lines
(Frost et al., 2001). Further investigation is needed to determine key components of this
potential mechanism of astrogliosis, including the subcellular localization of active caspases,
the identity of relevant transcription factors, and the relationships between these components
and regulation of specific factors including GS and FGF-2.
Several non-apoptotic functions of caspases are attributed to sublethal activation
(McLaughlin, 2004). Apoptotic or non-apoptotic consequences may occur due to differential
cleavage of substrate depending upon the level of caspase activation. In the current study,
low levels of caspase activation were observed in astrocytes following dB-cAMP and Aβ
apoptosis treatments in the absence of cell death, whereas high levels of caspase activation
and apoptosis were observed following staurosporine treatment. Additionally, apoptotic
inhibitors including survivin and heat shock protein 25/27 have been found to co-localize
with active caspase-3 in the nucleus of reactive astrocytes following brain injury, potentially
inhibiting apoptotic actions of caspases (Villapol et al., 2008).
3.3 Conclusions
In summary, these findings support a non-apoptotic role for caspase activation in the
regulation of astrogliosis. Growing evidence suggests that caspases exert dual roles in the
injured brain: facilitating apoptosis in severely injured neurons while also protecting neurons
as a consequence of increased expression of bioactive factors from reactive astrocytes.
Similarly, other signaling cascades also perform seemingly opposing functions, such as p38
MAPK, which is commonly activated following neuronal injury and can contribute to both
apoptosis and astrogliosis (Che et al., 2001a; Che et al., 2001b). Additional signaling
molecules implicated in astrogliosis, including ERK/MAPK (Mandell and VandenBerg,
1999; Mandell et al., 2001) and NF-κB (de Freitas et al., 2002; Perez-Otano et al., 1996)
also have diverse functions in the brain, such as differentiation, proliferation, and regulation
of apoptosis or cell survival pathways. Although the signaling cascades regulating induction
and maintenance of astrogliosis require additional definition, the current findings indicate
that caspases are significant contributors to the process.
4. EXPERIMENTAL PROCEDURE
4.1 Materials
Kainate, staurosporine, dBcAMP and other reagents (unless otherwise specified) were
purchased from Sigma-Aldrich (St Louis, MO). β-Amyloid 25–35 peptide (Aβ25–35) was
purchased from Bachem (Torrence, CA), solubilized in sterile, deionized water to 1 mM to
yield aggregated species, as previously described (Pike et al., 1995). Caspase inhibitors
(zVAD-fmk, DEVD-fmk, WEHD-fmk, AEVD-fmk, LEHD-fmk and LETD-fmk) were
purchased from Enzyme Systems (MP Biochemicals, Irvine, CA) and solubilized in DMSO.
4.2 Astrocyte cultures
Neonatal astrocytes cultures were generated from cerebral cortices of postnatal day 0–1
Sprague-Dawley rat pups using the method of McCarthy and de Vellis (1980) with
modifications as described previously (Pike et al., 1996b). In brief, cerebral cortices were
dissected and the tissue enzymatically and mechanically dissociated. The resultant cell
suspensions were plated into 75 cm2 flasks containing DMEM (Gibco, Invitrogen, Carlsbad,
CA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA)
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7. and maintained in a humidified incubator at 37° C with 5% CO2. Media was changed every
three days until the cultures reached confluence after 10–14 days in vitro. Once confluent,
culture flasks were shaken overnight at 280 RPM in an orbital shaker at 37° C, a procedure
that eliminates most non-astroglial cells. The percentage of microglial cells was <5% as
determined by postive immunoreactivity with the microglial marker Iba-1. Enriched
astroglia were re-plated at 12,000 cells/cm2 onto poly-lysine coated plates in DMEM/10%
FBS. The cultures were shifted to serum free medium 1–3 days prior to experimental use.
Adult astrocytes were cultured from hippocampi of 3 month-old adult female Sprague-
Dawley rats using the method of Schwartz and Wilson (1992). Dissected hippocampi were
enzymatically and mechanically dissociated, then plated into 50 cm2 flasks containing
DMEM/20% FBS and placed in a humidified incubator at 37° C with 5% CO2. Media was
changed every three days for 10–14 days and at least 15 separate astrocyte colonies were
detected. Cultures were then passaged once and grown to confluency. Confluent cultures
were shaken overnight at 37°C at 120 RPM on an orbital shaker to eliminate non-astroglial
cells. Enriched adult astroglial cultures were re-plated at 12,000 cells/cm2 onto poly-lysine
coated plates in DMEM/F12 (Gibco, Invitrogen, Carlsbad, CA) with 20% FBS. The cultures
were shifted to serum-free DMEM/F12 1–3 days prior to use in experiments.
4.3 Cell viability
Cell viability was assessed by two methods. First, numbers of non-viable cells were counted
following staining with the vital dyes calcein AM (live cells) and ethidium homodimer (dead
cells) (Molecular Probes; Invitrogen, Carlsbad, CA). Counts of non-viable cells (negative
for calcein AM, positive for ethidium homodimer) were made in four non-overlapping fields
per culture well (in a predetermined pattern maintained across all experiments) with each
condition represented by 3 separate wells. All experiments were repeated in at least 3
independent culture preparations. Second, viability was also quantified by assaying culture
media for levels of the intracellular enzyme lactate dehydrogenase, which is released from
dying cells with compromised membranes. Lactate dehydrogenase activity was assessed in
culture media via spectrophotometric assay as previously described (Koh and Choi, 1987).
Briefly, 0.5 mg/ml β-NADH in potassium phosphate buffer was incubated with the
treatment media at room temperature for 30 minutes. Next, the substrate pyruvate (12 mM)
was added and the plate read at 340 nm every 8 sec over a 5 min period using a Molecular
Devices spectrophotometer and data analyzed using SOFTmax PRO (Molecular Devices,
Sunnyvale, CA). Samples were assayed in triplicate and each condition assessed in four
independent experiments. For both methods, some culture wells were treated with
staurosporine (1 μM) as an internal standard for maximum cell death.
4.4 Western blot
Immunoblotting was performed as previously described (Pike et al., 1996a). In brief, equal
amounts of culture lysates were diluted into reducing sample buffer and electrophoresed at
constant 100V in 10% or 12% polyacrylamide gels using standard SDS-PAGE
methodology. Proteins were transferred onto 0.45 μm PVDF membranes (Millipore;
Bedford, MA). Membranes were incubated for 1 h in 3% bovine serum albumin to block
non-specific protein binding, then were probed with primary antibodies, including GFAP
(1:15000 dilution, DAKO, Carpinteria, CA), GS (1:1000 dilution) and FGF-2 (1:500
dilution, BD Biosciences, San Diego, CA). Caspase-3 was probed with an antibody specific
to the cleaved, active fragment (1:50 dilution, EMD Biosciences, Gibbstown, NJ). To
confirm equal amount of protein in all conditions, blots were stripped and re-probed with β-
tubulin (1:500 dilution, Chemicon, Temecula, CA). For data quantification, images from
scanned films were analyzed by NIH image 1.61 and relative protein quantities were
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8. normalized to control groups. Results from at least 3 independent experiments were
combined for statistical analysis.
4.5 Caspase activity assay
Total caspase activity was assessed in duplicate according to manufacturers’ instructions via
fluorometric assay (Calbiochem, Gibbstown, NJ) on cell suspensions collected through
mechanical scraping. Briefly, protein levels were assessed in an aliquot of cell suspensions
to ensure equal protein content in different treatment condition. After correcting for protein
content, cell suspension from each condition was incubated with FITC-conjugated caspase
substrate for 45 minutes. Cells were washed three times in wash buffer provided in the kit,
then resuspended and loaded onto a 96-well plate and read at emission wavelength of 345
nm and excitation wavelength of 548nm on a fluorometric plate reader (Molecular Devices).
4.6 Kainate lesion
Female Sprague-Dawley (3 months of age, N = 9/group) were anesthetized with sodium
pentobarbital (i.p., 50 mg/kg body weight). Kainate (1.5 μl of 0.5 mg/ml kainate in saline) or
sterile saline solution (1.5 μl) was injected bilaterally into the hippocampi using a Hamilton
syringe and stereotaxis (coordinates: -5.2 bregma, 4.5 lateral to the mid-line, 4.0 mm from
the surface of the cortex). Two weeks following kainate lesion, animals were sacrificed by
decapitation and the brains quickly harvested. The left hemisphere was immersion fixed for
48 hours in 4% paraformaldehyde/0.1 M PBS for use in immunohistochemical analyses. The
right hemisphere was immediately used to generate adult astrocytes cultures in an ex-vivo
paradigm of astrogliosis, as described above.
4.7 Immmunochemistry
Fixed hemi-brains were exhaustively sectioned (40 μm) in the horizontal plane using a
vibratome, and then subjected to immunohistochemistry using standard avidin-biotin
methodology, as previously described (Ramsden et al., 2003). For each brain, every tenth
section containing hippocampus was permeabilized in 0.02% Triton-X100 in 0.1 M tris-HCl
(pH 7.4), then incubated in 3% bovine serum albumin/0.1M tris followed by either NeuN
antibody (1:500 dilution; Chemicon, Temecula CA) or GFAP antibody (1:15,000 dilution;
ICN, Irvine, CA) for 24 hrs. Next, sections were incubated with either anti-mouse (for
NeuN) or anti-rabbit (for GFAP) biotinylated antibody (Vector Laboratories, Burlingame,
CA) for 1 hr followed by avidin-biotin-peroxidase complex (ABC, Vector Laboratories) for
1 hour. Immunostaining was visualized using 3,3′ diaminobenzidine (DAB, Vector
Laboratories). Sections were dehydrated and coverslipped with depex mounting medium.
For GFAP immunocytochemistry, astroglial cultures were fixed with 2% paraformaldehyde
and processed as described above using GFAP antibody (1:15000, DAKO, Carpinteria, CA).
4.8 Statistical analyses
Raw data were statistically assessed using one-way ANOVA followed, when appropriate, by
between group comparisons using Fisher Least Squares Difference test. A P value of < 0.05
was considered significant.
Acknowledgments
AMB is supported by the American-Australian Neurological Fellowship. This study was supported in part by NIH
grants AG15961 (CJP) and AG026572 (RD Brinton/CJP).
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9. Abbreviations
Aβ β-amyloid
dBcAMP dibutryl cyclic adenosine monophosphate
FGF-2 fibroblast growth factor-2
GFAP glial fibrillary acidic protein
GS glutamine synthetase
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13. Highlights
• Astrogliosis is associated with non-apoptotic activation of caspases
• Caspase inhibitors attenuate astrogliosis in neonatal astrocyte cultures
• Caspases contribute to astrogliosis in adult astrocyte cultures
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14. Figure 1. Reactivity observed in primary astrocytes treated with dB-cAMP or Aβ
(A) Vehicle-treated control (Ctl) neonatal astrocyte cultures show polygonal morphology. In
contrast, stellate morphology is observed in cultures treated with (B) 1 mM dBcAMP (dB)
or (C) 25 μM Aβ25–35. Representative western blots demonstrate increased expression of
(D) GS and (E) FGF-2 in cultures treated for 48 h with dBcAMP (dB) or Aβ relative to
vehicle-treated controls (Ctl).
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15. Figure 2. Increased caspase activity following treatment with reactive stimuli dB-cAMP or Aβ
Treatment of neonatal astrocytes cultures with 1 mM dB-cAMP (dB) or 25 μM Aβ25–35 for
48 h significantly increased (A) total caspase activity and (B) levels of the active caspase-3
fragment as assessed by western blot (inset: representative western blot). Cultures treated for
24 h with the apoptotic stimulus 1μM staurosporine (STS) demonstrated the greatest
increase in total caspase activity and levels of the active caspase-3 fragment. Data show
mean levels (+SEM) from ≥ 3 independent experiments. * P < 0.01 relative to vehicle-
treated control (Ctl).
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16. Figure 3. Absence of cell death following treatment with reactive stimuli db-cAMP or Aβ
(A) Viability of neonatal astrocytes cultures treated with vehicle (CTL), 1 mM dBcAMP or
25 μM Aβ25–35 for 4 d or 1 μM staurosporine (STS) for 24 h was assessed by staining with
the vital dyes calcein AM (green fluourescence; indicates live cells), and ethidium
homodimer (red fluorescence; indicates dead cells). Levels of cell death in treated astrocytes
cultures were quantified by (B) counts of cells with positive staining for ethidium
homodimer, and (C) measurement of LDH release into the culture medium. Data show mean
levels (+SEM) from ≥ 3 independent experiments. * P < 0.001 relative to vehicle-treated
control (Ctl).
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17. Figure 4. Broad-spectrum caspase inhibition attenuates up-regulation of GS and FGF-2
expression in response to reactive stimuli, dB-cAMP and Aβ, in astrocyte cultures
Neonatal astrocyte cultures were treated for 48 h with vehicle (Ctl), 1 mM dBcAMP (dB), or
25 μM Aβ25–35 in the absence (−) or presence (+) or 1 h pretreatment with 50 μM zVAD.
Following the experiment, cultures were processed for western blot to assess levels of GS
(A) and FGF-2 (C). β-Tubulin was assessed as to ensure equal protein loading. Relative
protein levels of GS (B) and FGF-2 (D) were determined by densitometric scanning of
western blots from ≥ 3 independent experiments. Data show mean values (+SEM) from each
condition expressed as a percentage of the vehicle-treated control group. * P < 0.05 relative
to matched (−) zVAD condition.
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18. Figure 5. Astrocyte stellation unaffected by broad spectrum capsase inhibition
Neonatal astrocytes cultures were pretreated for 1 h with vehicle (veh), 50 μM zVAD, or 10
nM thrombin followed by 48 h exposure to 1 mM dBcAMP (dB) or 25 μM Aβ25–35 and then
were processed for GFAP immunocytochemistry. (A) Representative bright field images of
GFAP immunostaining show that stellate morphology is attenuated by pretreatment with
thrombin but not zVAD. (B) Stellation was quantified from immunostained cultures by
determining mean counts of morphologically stellate cells (+SEM) from each condition (N =
3). * P < 0.05 relative to matched condition.
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19. Figure 6. Specific inhibition of caspases-3 and -11 attenuates GS and FGF-2 expression in
reactive astrocytes
Neonatal astrocytes cultures were pretreated for 1 h with increasing concentration of caspase
inhibitors (0, 1, 10, 30, 60, 100 μM) followed by 48 h exposure to 1 mM dBcAMP (dB). For
comparison, experiments included conditions with vehicle (Ctl) or 100 μM inhibitor (100) in
the absence of dBcAMP. At the conclusion of the experiment, cultures were processed for
western blots to assess levels of GS, FGF-2, and β-tubulin (internal control). Representative
blots are shown for astrocyte cultures pre-treated with (A) the caspase-3 inhibitor DEVD,
and (C) the caspase-11 inhibitor WEHD. Densitometric analysis of blots revealed significant
inhibition of both GS and FGF-2 expression in astrocyte cultures treated with (B) 10 – 100
μM DEVD, and (D) 30–100 μM WEHD. Data show mean values (± SEM) relative to
dBcAMP-treated cultures in the absence of caspase inhibitor. *P < 0.05 relative to condition
lacking inhibitor.
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20. Figure 7. GS and FGF-2 expression in reactive astrocytes unaffected by specific inhibition of
caspases-1, -6, -8 and -9
Neonatal astrocytes cultures were pretreated for 1 h with increasing concentration of caspase
inhibitors (Inh; 0, 1, 10, 30, 60, 100 μM) followed by 48 h exposure to 1 mM dBcAMP
(dB). For comparison, experiments included conditions with vehicle (Ctl) or 100 μM
inhibitor (100) in the absence of dBcAMP. At the conclusion of the experiment, cultures
were processed for western blots to assess levels of GS, FGF-2, β-tubulin (internal control).
Representative blots for (A) GS and (B) FGF-2 are shown for astrocyte cultures pre-treated
with specific inhibitors for caspase-1 (YVAD), caspase-6 (AEVD), caspase-8 (LETD) or
caspase-9 (LEHD). At non-specific concentrations (>50μM) AEVD and LEHD significantly
attenuated both GS and FGF-2 expression, while YVAD attenuated FGF-2 but not GS
expression. Data show mean values (± SEM) relative to dBcAMP-treated cultures in the
absence of caspase inhibitor.
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21. Figure 8. Kainate lesion results in neuronal loss and astrogliosis
Adult female rats were lesioned bilaterally by intra-hippocampal injection with kainate then
3 weeks later processed for immunohistochemistry. Labeling with the neuron-specific
antibody NeuN shows that the hippocampus CA3 pyramidal neuron layer (arrows) is intact
in vehicle-lesioned (Sham) animals (A) but largely degenerated in kainate-lesioned
(Kainate) rats (B). Astrocyte staining with GFAP antibody shows that, in comparison to
sham-lesioned rats (C), lesioned rats display robust labeling throughout hippocampus (D)
that is characterized by robust stellation (magnified inset). Images show representative
findings from sham-lesioned (N = 4) and kainate-lesioned (N = 5) animals.
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22. Figure 9. Evidence of non-apoptotic caspase activation in an ex vivo model of astrogliosis
Astrocytes cultured from adult sham-lesioned (Sham) and kainate-lesioned (Kainate) rats
were characterized for morphology, caspase activity and viability. GFAP immunostaining of
cultures revealed similar morphological appearance with little stellation in hippocampal
astrocyte cultures derived from (A) sham-lesioned (N = 4) and (B) kainate-lesioned (N = 5)
rats. (C) Astrocyte cultures from sham-lesioned animals (Sham) and kainate-lesioned
animals (Kainate) did not exhibit significant differences in LDH release. For comparison,
cultures from sham-lesioned animals treated for 24 h with 1 μM staurosporine (STS) showed
a significant increase in LDH release. (D) Total caspase activity in cultures was assessed in
the presence (solid bars) and absence (open bars) of the general caspase inhibitor zVAD (50
μM). Caspase activity was significantly elevated both in cultures from kainate-lesioned
animals and in cultures of sham-lesioned animals treated for 24 h with 1 μM staurosporine.
Data show mean values (+SEM) from 4–5 independent culture preparations. * P < 0.05
relative to Sham condition. (E) Representative western blots show relative levels active
caspase-3 expression from sham- and kainate-lesioned astrocyte cultures in the presence (+)
and absence (−) of 50 μM zVAD treatment and following treatment with 1μM staurosporine
(STS) for 1 h or 24 h. Bots were stripped and reprobed β-tubulin as an internal control for
equal protein.
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23. Figure 10. Caspase inhibition attenuates up-regulation of GS and FGF-2 expression in an ex vivo
model of astrogliosis
Astrocyte cultures from sham- and kainate-lesioned rats were treated for 48 h with vehicle
(−) or 50 μM general caspase inhibitor zVAD (+) and then processed for western blot.
Representative western blots show increased (A) GS and (C) FGF-2 expression in cultures
from kainate-lesioned (KA 1, KA 2) rats compared to sham-lesioned controls (Sham 1,
Sham 2). Densitometric analysis of western blots revealed zVAD treatment (+zVAD; solid
bars) significantly decreased (B) GS and (D) FGF-2 expression in astrocyte cultures from
Kainate-lesioned rats (Sham, N = 4; Kainate, N = 5). * P < 0.05 relative to matched
condition.
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